The present invention is in the field of tumor biology. More specifically, the present invention relates to use in vivo of dominant negative deletion mutants of c-jun in methods for the treatment and prevention of cancer.
Chemical carcinogenesis is a multistep process that includes initiation, promotion and progression (Boutwell, R. K. (1964) Prog. Exp. Tumor Res., 4: 207-250; Drinkwater, N. R. (1989) In: Genes and Signal Transduction in Multistage Carcinogenesis, (ed) Colburn, N. H., Marcel Dekker, Inc., New York, pp. 3-17); Dong, Z. et al. (1990) Cancer Invest., 8: 523-533; Weinstein, I. B. (1988) Cancer Res., 48: 4135-4143) with the rate-limiting steps in multistage carcinogenesis occurring during the promotion and progression phases. Numerous studies have shown that tumor promotion is a long term process that is partially reversible and that requires chronic exposure to tumor promoter. In vivo studies in the mouse have also found susceptibility to promotion of neoplastic transformation to be inheritable and genetically controlled (Drinkwater, N. R. (1989)).
In recent years, numerous cellular oncogenes have been implicated in the transactivation of genes implicated in cellular growth and differentiation. One such cellular oncogene, c-jun, encodes a 39-kDa nuclear phosphoprotein c-jun which is a component of the heterodimeric transcriptional activator AP-1 along with the c-Fos proto-oncoprotein. (Karin, M. et al. (1991) Biochem. Biophys. Acta., 1072: 129-157; Vogt, P. K. et al (1990) Adv. Cancer Res., 55: 1-35; Curran, T. et al. (1988) Cell., 55: 395-397). The AP-1 complex has been demonstrated to transcriptionally activate genes that contain the sequence TGAg/cTCA, referred to as an AP-1 binding site or TPA-responsive element (TRE), in their promoters (Karin, M. and Angel, P. (1991) Biochim. Biophys. Acta., 1072: 129-157, Vogt, P. K. et al (1990) Adv. Cancer Res., 55: 1-35, Curran, T. et al (1988) Cell., 55: 395-397). In addition to the induction of AP-1 activity by TPA treatment, AP-1 activity has also been shown to be blocked by retinoic acid (RA) and glucocorticoids (Schule, R. et al. (1990) Cell, 62: 1217-1226; Schule, R. et al. (1991) Proc. Natl. Acad. Sci. USA., 88: 6092-6096; Nicholson, R. C. et al. (1990) EMBO. J., 9: 4443-4454; Jonat, C. et al. (1990) Cell, 62: 1189-1204; Yang, N. et al. (1991) Proc. Natl. Acad. Sci. USA., 88: 3559-3563). Of interest, several genes that may be involved in tumor promotion or progression have been shown to respond to AP-1 including the genes for the metalloproteinases collagenase and stromelysin (transin) (Matrisian, L. M. et al. (1991) Am. J. Medic. Sci., 302: 157-162, Angel, P., et al. (1987) Mol. Cell. Biol., 7: 2256-2266, Honoki, K. et al. (1992) Mol. Carcinogenesis., 6: 122-128).
Analysis of the c-jun and c-fos phoshoproteins has demonstrated that they each contain basic DNA-binding domains, a leucine zipper domain involved in dimerization, and transactivation domains involved in transcriptional regulation (Alani et al. (1991) Mol. Cell Biol., 11:6286-6295). The c-jun protein, through protein-protein interactions within the leucine zipper domain, is able to dimerize with itself (Smeal, T. et al. (1989) Genes Dev., 3: 2091-2100; Turner, R. et al. (1989) Science, 243: 1689-1694) or with other leucine zipper-containing proteins such as other c-jun family members (JunB or JunD) (Halazonetis T. D. et al. (1988) Cell, 55: (917-924), fos family members (c-Fos, FosB, Fra-1 or Fra-2) (Smeal, T. et al. (1989); Turner, R. et al. (1989); and Zerial, M. et al. (1989) Embo J., 8: 805-813), the cyclic AMP-responsive element binding protein (CREB) (Sassone-Corsi, P. et al. (1990) Oncogene, 5: 427-431) and potentially other yet unidentified leucine zipper proteins.
Recent studies of the c-jun proto-oncogene have revealed that the regions critical for transcriptional activation include the dimerization domain, the DNA-binding domain, and an area within the amino terminal half of the c-jun protein that functions as a transcriptional activation domain (Hirai, S. et al. (1990) Oncogene, 5: 39-46; Baichwal, V. R. et al. (1990) Cell, 63: 815-825; and Abate, C. et al. (1991) Mol. Cell. Biol., 11: 3624-3632. Studies by Alani et al. (1991) Mol. Cell. Biol., 11: 6286-6295) demonstrated that regions of c-jun required for transcriptional activation are the same as those required for c-jun/ras-induced transformation of rat embryo cells thereby suggesting that c-jun might transform cells by altering gene expression through dysregulated DNA transcription. More recently, Brown et al. ((1993) Oncogene, 8: 877-886)) have shown that a dominant negative c-jun mutant which specifically blocked AP-1 activity also blocked H-ras plus c-jun induced cellular co-transformation. In addition, extensive evidence showing that endogenous hormones and growth factors play a major role in tumor promotion in human carcinogenesis suggests that tumor promoter-induced transformation is an important step in how humans actually get cancer. Thus, identification of dominant negative deletion mutants of c-jun which are capable of suppressing tumor promoter-induced neoplastic transformation might be of great utility in the prevention of carcinogenesis in vivo.
The present invention relates to a method of preventing carcinogenesis in mammals comprising: administering to a mammal at least one deletion mutant of c-jun inhibitory to tumor promoter-induced neoplastic transformation in an amount effective to prevent carcinogenesis.
The invention also provides pharmaceutical compositions for the prevention of carcinogenesis in mammals where said pharmaceutical compositions comprise at least one deletion mutant of c-jun inhibitory to tumor promoter-induced neoplastic transformation in a suitable diluent or carrier.
The invention further relates to a method for treating cancer comprising: administering to a mammal having cancer at least one deletion mutant of c-jun inhibitory to tumor promoter-induced neoplastic transformation in a therapeutically effective amount.
The deletion mutants of the present invention may be a protein or a nucleic acid sequence encoding the protein. It is therefore an object of the present invention to provide nucleic acid sequences capable of directing production of deletion mutants of c-jun in a host organism. Such nucleic acid sequences may be obtained by PCR amplification of cloned c-jun DNA or by restriction digestion and linker ligation of cloned c-jun DNA in a plasmid. For purposes of this application, nucleic acid sequence refers to RNA, DNA, cDNA or any synthetic variant thereof which encodes a deletion mutant of c-jun protein.
It is also an object of the present invention to provide deletion mutants of c-jun where said deletion mutants are proteins inhibitory to tumor promoter-induced neoplastic transformation.
The present invention also relates to a method for determining whether a tumor promoter induces transformation via a c-jun dependent pathway, said method comprising: (a) transfecting a cell line transformable by treatment with said tumor promoter with nucleic acid sequence encoding a deletion mutant of c-jun inhibitory to tumor promoter-induced neoplastic transformation; (b) treating transfected and untransfected cells with said tumor promoter in an amount effective to stimulate neoplastic transformation of said cells; and (c) measuring transformation of said transfected and untransfected cells by a suitable transformation assay.